Identifying the functions of a family of nuclear RNA binding proteins that switch expression between somatic and meiotic cells

研究目的。脊椎动物基因在RNA结合蛋白的引导下产生广泛加工的RNA，这些RNA结合蛋白通常由多基因家族编码。然而，这些家族中蛋白质的个体功能往往不被很好地理解。本研究的目的是提供RBMX基因家族的第一个全球分析，该基因家族在所有哺乳动物中都是保守的，在所有脊椎动物中都有成员。RBMX家族的核RNA结合蛋白由RBMX、hnRNP GT和RBMY蛋白组成，它很有趣，因为在体细胞（遍布全身）和减数分裂细胞（最终产生精子）之间存在基因表达转换。小鼠基因敲除的初步数据表明，这些基因表达开关是至关重要的，但为什么会这样，或者单个RBMX家族蛋白的作用在很大程度上仍然未知。及时性和物有所值。我们已经建立了条件，在这种条件下，我们可以耗尽体细胞中的RBMX蛋白，并诱导一种内源性目标转录物的RNA加工变化，我们将能够扩大到更全面的分析。我们还制作了一个新的小鼠模型，从中我们可以删除高度保守的Hnrnpgt基因。尽管这些基因敲除小鼠在其他方面是健康的，但Hnrnpgt缺失会导致生殖细胞在减数分裂的关键阶段二倍体期停滞。RBMX敲低和hnRNP GT缺失这两项进展的结合，为我们了解RBMX和hnRNP GT蛋白活性如何协调控制核基因表达途径提供了机会。目的1将确定体细胞中RBMX蛋白调控的靶基因和通路，以及RBMX功能的机制。目的2将寻找hnRNP GT蛋白在减数分裂中调控的基因和途径，并试图确定这些基因和途径与体细胞中RBMX调控的基因和途径是否相同或不同。虽然我们不知道没有hnRNP GT蛋白的哪些途径会被破坏，但我们可以从Hnrnpgt缺失小鼠的详细表型中做出强有力的预测。HnRNP - GT null生殖细胞在减数分裂时处于二倍体期，这是成对染色体刚刚开始分离的重要阶段。我们的初步数据显示，在hnRNP - GT阴性睾丸中，减数分裂染色体异常扩散，这表明至少hnRNP - GT的一些靶点会控制减数分裂染色体结构。有趣的是，RBMX也与染色体结构有关，但机制尚不清楚。目的3将进一步利用我们的新小鼠模型来确定hnRNP GT在减数分裂后是否具有重要功能，在此期间RBMX仍然关闭。目的4将测试我们发现的由小鼠RBMX和hnRNP GT控制的RNA加工途径是否在人类中保守，并在不育男性中被错误调控。综上所述，这些目标将剖析这个高度保守的RNA结合蛋白家族中的个体功能，以及个体蛋白之间的表达开关如何控制基因表达的全局模式。特别是，我们将测试RBMX和hnRNP GT表达之间的转换如何促进全球剪接程序的变化。结果。这项研究的预期结果将是更好地了解核RNA结合蛋白家族如何控制发育途径。这样的计划可能对全身都很重要。该项目将揭示RBMX蛋白对人类健康的重要性。RBMX家族基因与人类男性不育、大脑和肌肉发育以及癌症有关，但人们对其了解甚少。这将是对这一RNA结合蛋白家族的首次全面分析。这个项目的受益者将是对基因表达和发育感兴趣的科学家、我们培养的科学家和学生、不育男性和我们接触的公众成员。

PURPOSE OF RESEARCH. Vertebrate genes make extensively processed RNAs guided by RNA binding proteins that are often encoded by multigene families. However, the individual functions of proteins within these families are frequently not well understood. The purpose of this research is to provide the first global analysis of the RBMX gene family which is conserved in all mammals, and has members conserved in all vertebrates. The RBMX family of nuclear RNA binding proteins consists of RBMX, hnRNP GT and RBMY proteins, and is interesting since there are gene expression switches between somatic cells (found all over the body) and meiotic cells (that eventually give rise to sperm). Preliminary mouse knockout data indicates that these gene expression switches are critical, but why this is or what individual RBMX family proteins do is still largely unknown.TIMELINESS AND VALUE FOR MONEY. We have already established conditions in which we can deplete the RBMX protein in somatic cells, and induce changes in RNA processing of one endogenous target transcript, which we will be able to scale up to a more global analysis. We have also made a new mouse model, from which we can delete the highly conserved Hnrnpgt gene. Although these knockout mice are otherwise healthy, Hnrnpgt deletion causes germ cells to arrest during meiosis at a crucial stage called diplotene. The combination of these two advances, RBMX knockdown and hnRNP GT deletion, provide us the opportunity to understand how the coordination of RBMX and hnRNP GT protein activity controls pathways of nuclear gene expression.OBJECTIVES. Objective 1 will identify target genes and pathways controlled by the RBMX protein in somatic cells, and mechanisms of RBMX function. Objective 2 will search for genes and pathways regulated by hnRNP GT protein in meiosis, and seek to determine if these are the same or different to those regulated by RBMX in somatic cells. Although we don't know what pathways are disrupted without hnRNP GT protein, we can make a strong prediction from the detailed phenotype of our Hnrnpgt null mice. HnRNP GT null germ cells arrest during meiosis at diplotene, an important stage where the paired chromosomes are just starting to separate. Our preliminary data show that meiotic chromosomes are abnormally spread out in Hnrnpgt null testes, suggesting at least some of the targets of hnRNP GT will control meiotic chromosome structure. Interestingly RBMX has also been connected to chromosome structure, but the mechanism is not known. Objective 3 will take further advantage of our new mouse model to determine if hnRNP GT has important functions after meiosis, during which time RBMX is still turned off. Objective 4 will test if the RNA processing pathways we find to be controlled by mouse RBMX and hnRNP GT are conserved in humans, and mis-regulated in infertile men. Taken together, these objectives will dissect individual functions within this highly conserved family of RNA binding proteins, and how switches in expression between individual proteins control global patterns of gene expression. In particular, we will test how the switch between RBMX and hnRNP GT expression contributes to changes in global splicing programmes. OUTCOMES. The expected outcomes from this research will be a better understanding of how families of nuclear RNA binding proteins function to control pathways of development. Such programmes are likely important all over the body. This project will shed new light on how the RBMX proteins are important to human health. RBMX family genes are implicated in human male infertility, brain and muscle development and cancer, but still poorly understood. This would be the first comprehensive analysis of this family of RNA binding proteins. The beneficiaries of this project will be scientists interested in gene expression and development, scientists and students that we train, infertile men and members of the public with whom we engage.